Creation of libraries of droplets and related species

ABSTRACT

The present invention is generally related to systems and methods for producing a plurality of droplets. The droplets may contain varying species, e.g., for use as a library. In some cases, the fluidic droplets may be rigidified to form rigidified droplets (e.g., gel droplets). In certain embodiments, the droplets may undergo a phase change (e.g., from rigidified droplets to fluidized droplets), as discussed more herein. In some cases, a species may be added internally to a droplet by exposing the droplet to a fluid comprising a plurality of species.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/098,674, filed Sep. 19, 2008, entitled “Creationof Libraries of Droplets and Related Species,” by Weitz, et al., hereinincorporated by reference.

GOVERNMENT FUNDING

This invention was made with government support under DMR-0602684awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF INVENTION

The present invention is generally related to systems and methods forproducing a plurality of droplets. The droplets may contain varyingspecies, e.g., for use as a library. The droplets may be rigidifiedand/or fluidized, in some cases.

BACKGROUND

Various technologies have been developed that allow production of“libraries” of synthetic compounds (e.g., pharmaceutical agents,inhibitors, etc.) or nucleic acid probes, etc. A library of syntheticcompounds may be used to screen for a target analyte molecule (e.g., anenzymes, an antibody, etc.) having a desired activity. Although currentlibraries are valuable, commercially-available resources useful in thesearch to identify new compounds with desirable activities, there arepractical difficulties (e.g., cost, time, equipment, etc.) associatedwith the preparation, storage, and use of current technologies.

SUMMARY OF THE INVENTION

The present invention is generally related to systems and methods forproducing a plurality of droplets. The droplets may be rigidified and/orfluidized, in some cases. The subject matter of the present inventioninvolves, in some cases, interrelated products, alternative solutions toa particular problem, and/or a plurality of different uses of one ormore systems and/or articles.

In one aspect, the invention is directed to a method. According to afirst set of embodiments, the method includes acts of providing aplurality of droplets, each of the plurality of droplets comprising afirst fluid and being surrounded by a second fluid, wherein the firstfluid and the second fluid are substantially immiscible, causing atleast some of the plurality of droplets to undergo a phase change toform a plurality of rigidified droplets, exposing the plurality ofrigidified droplets to a third fluid, wherein the third fluid issubstantially miscible in the first fluid, and adding at least one firstspecies internally to at least some of the rigidified droplets.

In one set of embodiments, the method includes acts of providing aplurality of groups of rigidified droplets, each of the groups ofrigidified droplets comprising a first fluid and having substantiallythe same composition as the other groups of rigidified droplets butcontaining a distinguishable species with respect to the other groups ofrigidified droplets, forming a suspension comprising at least onerigidified droplet from each of the groups of rigidified droplets,exposing at least some of the rigidified droplets of the suspension to asecond fluid, wherein the first fluid and the second fluid aresubstantially immiscible, and fluidizing at least some of the rigidifieddroplets to form a plurality of fluidized droplets, wherein theplurality of fluidized droplets are substantially immiscible in thesecond fluid.

In another set of embodiments, the method includes acts of providing afirst plurality of rigidified droplets, each rigidified droplet beingsurrounded by a second fluid, the rigidified droplets being formed froma fluidic droplet comprising a first fluid, wherein the second fluid issubstantially immiscible in the first fluid, exposing the firstplurality of rigidified droplets to a third fluid, wherein the thirdfluid is substantially miscible in the first fluid, adding at least onefirst species internally to at least some of the first plurality ofrigidified droplets to form a first group of species-containingdroplets, repeating the above acts with a second plurality of rigidifieddroplets and a second species to form a second group ofspecies-containing droplets, and forming a suspension comprising atleast some droplets of the first and second groups of species-containingdroplets.

In still another set of embodiments, the method includes the acts ofproviding a plurality of droplets, each of the plurality of dropletscomprising a first fluid and being surrounded by a second fluid, whereinthe first fluid and the second fluid are substantially immiscible,causing at least some of the plurality of microfluidic droplets toundergo a phase change to form a plurality of rigidified droplets,exposing the plurality of rigidified droplets to a third fluid, whereinthe third fluid is substantially miscible in the first fluid, separatingthe plurality of rigidified fluidic droplets into at least a first groupof rigidified droplets and a second group of rigidified droplets, addingat least one first species internally to at least some of the rigidifieddroplets of the first group and at least one second species internallyto at least some of the rigidified droplets from the second group,wherein the second species is distinguishable from the first species,forming a suspension comprising at least one rigidified droplet fromeach of the groups of rigidified droplets, fluidizing at least some ofthe rigidified droplets to form a plurality of fluidized droplets, andforming a plurality of microfluidic droplets, wherein each microfluidicdroplet comprises at least one rigidified droplet.

In still yet another set of embodiments, the method includes the acts ofproviding a first plurality of rigidified droplets, immobilizing atleast one first species internally with respect to at least some of thefirst plurality of rigidified droplets to form a first group ofspecies-containing droplets, repeating the above acts with a secondplurality of rigidified droplets and a second species to form a secondgroup of species-containing droplets, and forming a suspensioncomprising at least some droplets of the first and second groups ofspecies-containing droplets.

In another aspect, the invention is directed to a composition. In somecases, the composition comprises a container comprising at least eightmutually distinguishable rigidified droplets, each having substantiallythe same composition but containing a distinguishable species withrespect to the other rigidified droplets.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 depicts a method for forming a suspension comprising two groupsof droplets and for fusing the suspension of droplets with a pluralityof analyte droplets, according to some embodiments of the presentinvention.

FIGS. 2A and 2B depict non-limiting embodiments of the exchange offluids that may occur between a droplet and the fluid substantiallysurrounding the droplets, according to some embodiments of the presentinvention.

FIGS. 3A and 3B illustrate the fusing of an analyte droplet with afluidized droplet or a rigidified droplet, respectively, to form amicrofluidic droplet, according to some embodiments of the presentinvention.

FIG. 3C illustrates the formation of a microfluidic droplet comprising aspecies and/or a target analyte molecule.

FIG. 4 illustrates a method for forming a plurality of rigidifieddroplets according to one embodiment of the present invention.

FIG. 5 depicts a method for forming a plurality of fluidized dropletscomprising distinguishable species, according to some embodiments.

FIGS. 6A-6C show various methods for exposing a droplet of the presentinvention to a fluid.

FIGS. 7A-7F show non-limiting examples of nucleic acid probes.

FIG. 8 illustrates a method for forming a plurality of rigidifieddroplets according to one embodiment of the present invention.

FIG. 9A shows the formation of a plurality of droplets comprising DNAoligonucleotides, according to one embodiment.

FIG. 9B shows the plurality of droplets formed in FIG. 9A followingrigidification and exposure to water, according to one embodiment.

FIG. 9C shows a fluorescent microscope image of a first group orrigidified droplets comprising a first DNA oligonucleotide and a secondgroup of rigidified droplets comprising a second DNA oligonucleotide,respectively, and which have been visualized by exposure to a twodistinguishable labeled oligonucleotides, according to one embodiment ofthe present invention.

DETAILED DESCRIPTION

The present invention is generally related to systems and methods forproducing a plurality of droplets. The droplets may contain varyingspecies, e.g., for use as a library. In some cases, the fluidic dropletsmay be rigidified to form rigidified droplets (e.g., gel droplets). Thedroplets may undergo a phase change (e.g., from rigidified droplets tofluidized droplets), as discussed more herein. In some cases, a speciesmay be added internally to a droplet, such as a rigidified droplet byexposing the droplet to a fluid comprising the species. Afterintroduction, the species may be contained within the droplet, forinstance, reacted within the droplet, and/or the fluid surrounding thedroplet may be replaced by a fluid that is relatively unfavorable to thespecies.

The present invention, in some embodiments, provides methods for forminga composition comprising a plurality of droplets, where the droplets maybe fluidic or rigid. In some cases, the method provides a plurality ofdroplets containing a first fluid and being substantially surrounded bya second fluid, where the first fluid and the second fluid aresubstantially immiscible (e.g., droplets containing an aqueous-basedliquid being substantially surrounded by an oil-based liquid). At leastsome of the droplets may be caused to undergo a phase change to form aplurality of rigidified droplets (e.g., a plurality of gel droplets).The gel droplets may be exposed to a third fluid which is substantiallymiscible with the first fluid (e.g., an aqueous-based liquid) and therigidified droplets may be separated into at least a first group ofrigidified droplets and a second group of rigidified droplets. At leastone first species may be added internally to some of the first group ofrigidified droplets and at least one second species may be addedinternally to the second group of rigidified droplets, where the firstspecies and the second species are distinguishable with respect to theother species. A suspension may be formed comprising at least one of thefirst group of species-containing droplet and at least one of the secondgroup of species-containing droplets. In some cases, the suspension ofrigidified species- containing droplets may be fluidized (e.g., phasechange from a gel to a liquid) and a plurality of fluidized droplets maybe formed. In certain embodiments, a plurality of microfluidic dropletsis formed (e.g., by fusing the rigidified droplet with another fluidicdroplet) which each comprise at least one fluidized droplet, and in somecases, as least one target analyte molecule.

In some embodiments, the invention provides systems and methods forforming a plurality of droplets, where the plurality of dropletscomprises various groups of droplets containing at least onedistinguishable species with respect to other groups. For example, theplurality of droplets may comprise a first group of droplets and asecond group of droplets, where each of the first group of dropletscontains a first species and each of the second group of dropletscontains a second species. The plurality of droplets can be formed byproviding a plurality of rigidified droplets (e.g., gel droplets)containing a first fluid, substantially surrounded by a second fluid.The plurality of rigidified droplets may be exposed to a third fluid(which may be substantially miscible with the first fluid), and at leastone species may be added internally to each droplet.

The addition of a species to a droplet may be accomplished using anysuitable technique. In one embodiment, the species is added to thedroplet by allowing the species to diffuse from the fluid substantiallysurrounding the droplet into fluid contained within the droplet. Thismay be repeated for a second group of rigidified droplets and a secondspecies. In some cases, at least some of the first group of rigidifieddroplets and the second group of rigidified droplets may be combinedwithin a common suspension comprising the first and second droplets.

A non-limiting example of one embodiment of the invention is depicted isFIG. 1. In this figure, a plurality of droplets 4 comprising a firstfluid are substantially surrounded by second fluid 2. In some cases, thefirst fluid and the second fluid may be substantially immiscible suchthat the plurality of droplets 4 do not coalesce or dissolve with secondfluid 2. However, in other cases, as discussed below, the first fluidand the second fluid need not be substantially immiscible. The pluralityof droplets 2, in FIG. 1, is caused to undergo a phase change to form aplurality of rigidified droplets 6, as indicated by arrow 3. The phasechange may be induced, for example, by altering the temperature of thedroplets, by a chemical reaction, or the like. For instance, thedroplets may comprise a material such as agarose, which forms a gel whenthe polymer is cooled to a temperature below its gelling temperature.

In FIG. 1, the plurality of rigidified droplets 6 is then exposed to athird fluid 8, as indicated by arrow 5. Third fluid 8 may besubstantially immiscible with second fluid 2 and/or substantiallymiscible with the first fluid, although in some cases, as discussedbelow, these are not requirements. In some cases, for instance, if theplurality of droplets 4 were exposed to third fluid 8 before the phasechange described above, droplets 4 may coalesce with third fluid 8, forinstance if droplets 4 were aqueous-based, second fluid 2 was oil-based,and third fluid 8 was also aqueous-based. Accordingly, by rigidifyingdroplets 4, coalescence with third fluid 8 may be avoided or at leastreduced in some embodiments.

In some cases, the rigidified droplets are exposed to a variety ofconditions. For instance, the rigidified droplets may be exposed tovarious species, environmental conditions, or the like. In this example,the rigidified droplets have substantially the same composition, whichmay be useful in reducing differences due to the production of thedroplets themselves, as opposed to the varying conditions the dropletswere subsequently exposed to. For instance, referring again to FIG. 1,the plurality of rigidified droplets may be separated into a first group10 of rigidified droplets 14 and a second group 12 of rigidified droplet16, as indicated by arrow 7. As shown in FIG. 1, the first group andsecond group are divided by a partition within the same vessel; however,in other cases, the groups may be contained within separate vessels, orthe groups may be separate aliquots or samples, etc. The droplets mayalso be separated into more than two groups in other embodiments, asdiscussed below; only two groups are presented in FIG. 1 for reasons ofclarity. In this figure, at least one first species 18 is introducedinto at least some of first group 10 of rigidified droplets 14 to form afirst group 26 of species-containing droplets 22, as follows. Firstspecies 18 may be present in third fluid 8 (for example, added to thirdfluid 8 after separation of first group 10), and is allowed to associatewith or be contained internally in the droplet, e.g., via diffusion,chemical reaction, etc. Next, the suspension of droplets may be exposedto a fourth fluid 38. The fourth fluid may or may not be the same fluidas the second fluid, and in some cases, the fourth fluid may besubstantially immiscible with the first fluid. In some cases, fourthfluid 32 is chosen such that species 22 is not able to substantiallyleave droplets 14 to enter fourth fluid 32 (e.g., due to differences insolubility). However, in other cases, e.g., when species 22 ischemically reacted with droplet 14, the fourth fluid need not be onewhich discourages species 22 from leaving droplets 14, nor be one whichis substantially immiscible with the first fluid. Thus, by using anapproach such as the one discussed above, first species 18 may beintroduced into rigidified droplets 14 to form a first group 26 ofspecies-containing droplets 22. This process can also be repeated forthe second group 12 of rigidified droplets 16 (e.g., at least one secondspecies 20 may be added to at least some of the second group 12 ofrigidified droplets 16 to form a second group 28 of species-containingdroplets 24).

In some cases, droplets from one or more of these groups may be combinedtogether, e.g., to form a library. As an example, at least onerigidified droplet from the first group and at least one rigidifieddroplet for the second group may be combined to form a suspension ofrigidified droplets 30, as indicated by arrow 11. Of course, thisprocess can be repeated any number of times, e.g., 3, 4, or more timeswith different species, e.g., to form a library of droplets containingvarying species.

Optionally, the rigidified droplets 30 (which may contain species suchas first species 18 and/or second species 20) may be fluidized to formfluidized droplets 32, as indicated by arrow 13 in FIG. 1. The dropletsmay be fluidized, for example, by causing the droplets to undergo aphase change, e.g., by the temperature of the droplets, or by a chemicalreaction. For instance, rigidified droplets containing agarose may beheated to a temperature above its gelling temperature.

The rigidified and/or the fluidized droplets may then be manipulatedusing any suitable technique, depending on the application. Forinstance, the droplets may be identified, sorted, separated, split,fused or coalesced, mixed, charged, sensed, determined, etc., usingvarious systems and methods such as those disclosed in U.S. patentapplication Ser. No. 11/024,228, filed Dec. 28, 2004, entitled “Methodand Apparatus for Fluid Dispersion,” by Stone, et al., published as U.S.Patent Application Publication No. 2005/0172476 on Aug. 11, 2005; U.S.patent application Ser. No. 11/246,911, filed Oct. 7, 2005, entitled“Formation and Control of Fluidic Species,” by Link, et al., publishedas U.S. Patent Application Publication No. 2006/0163385 on Jul. 27,2006; U.S. patent application Ser. No. 11/885,306, filed Aug. 29, 2007,entitled “Method and Apparatus for Forming Multiple Emulsions,” byWeitz, et al.; or U.S. patent application Ser. No. 11/360,845, filedFeb. 23, 2006, entitled “Electronic Control of Fluidic Species,” byLink, et al., published as U.S. Patent Application Publication No.2007/0003442 on Jan. 4, 2007, each incorporated herein by reference.

As an example, the fluidized droplets shown in the example of FIG. 1 maybe fused with droplets 40. As shown in FIG. 1, droplets 34 and droplets36 may be combined to form a population of droplets 32 (some of whichcontain first species 18 and some of which contain second species 20,etc.), and the population of droplets are then fused with a plurality ofdroplets 40, as indicated by arrow 15. As a particular example, in someinstances, each of the plurality of droplets 40 comprises at least oneanalyte molecule 44 that one of first species 18 and second species 20may be able to interact with. In this example, each of the fused fluidicdroplets 48, formed after fusing each of droplets 40 with one ofdroplets 18 or 20, will comprise at least one analyte molecule 44 and atleast one of first species 18 or second species 20.

The plurality of microfluidic droplets may be analyzed, using a varietyof techniques, to determined the at least one analyte. Examples of suchtechniques include, but are not limited to, those described in U.S.patent application Ser. No. 11/024,228, filed Dec. 28, 2004, entitled“Method and Apparatus for Fluid Dispersion,” by Stone, et al., publishedas U.S. Patent Application Publication No. 2005/0172476 on Aug. 11,2005; U.S. patent application Ser. No. 11/246,911, filed Oct. 7, 2005,entitled “Formation and Control of Fluidic Species,” by Link, et al.,published as U.S. Patent Application Publication No. 2006/0163385 onJuly 27, 2006; U.S. patent application Ser. No. 11/885,306, filed Aug.29, 2007, entitled “Method and Apparatus for Forming MultipleEmulsions,” by Weitz, et al.; or U.S. patent application Ser. No.11/360,845, filed Feb. 23, 2006, entitled “Electronic Control of FluidicSpecies,” by Link, et al., published as U.S. Patent ApplicationPublication No. 2007/0003442 on Jan. 4, 2007, each incorporated hereinby reference.

It should be understood that the present invention is not limited toonly the methods described above. In various aspects, various speciescan be loaded into fluidic droplets, e.g., to form a library of species,and the fluidic droplets can be made rigid or made fluid, for instance,to facilitate loading of species within the droplets, or to manipulatethe species contained within the droplets. Accordingly, it should benoted that any combination of the above steps and/or additional stepsmay be used in the present invention, as discussed herein. For example,a plurality of rigidified droplets may be fused with a plurality ofmicrofluidic droplets without the step of fluidizing the plurality ofrigidified droplets. As another example, each group of rigidifieddroplets may also comprise at least one distinguishable identificationelement. As yet another example, the plurality of rigidified dropletsmay be separated into at least about 4096 groups. Each of the stepsand/or additional steps are discussed in detail herein, as well asnumerous examples of the combination of steps that may be performed.

In some embodiments of the present invention, a droplet, such as amicrofluidic droplet, may be caused to undergo a phase change from afirst phase to a second phase, which as mentioned above, may facilitatethe loading of a species within the droplet and the like. Non-limitingexamples of phases include solid phase, gel phase, liquid phase, gasphase, and the like. For example, a droplet may undergo a phase changefrom substantially liquid (e.g., the droplet is mostly in a liquidphase) to substantially solid (e.g., the droplet is mostly in a solidphase), or from substantially liquid to a substantially gel phase. It isimportant to note, however, that a phase change does not necessarilyneed to be a full change between two phases. For instance, a liquiddroplet may be at least partially rigidified to form a rigidifieddroplet or vice versa.

A “rigidified droplet,” as used herein, is a droplet that is in a statethat is not fluid, e.g., the droplet is not in a liquid or gaseousstate. For example, a rigidified droplet may be a solid droplet (e.g., aparticle), a gel droplet, and the like. Materials that may be used toproduce a rigidified or fluidized droplets, as well as methods to inducea phase change of a droplet are discussed more herein. As mentioned, insome cases, a rigidified droplet can be produced by causing a phasechange in a fluidic droplet. Non-limiting examples of phase changes of adroplet that is rigidified include substantially gas phase tosubstantially solid phase, substantially gas phase to substantially gelphase, substantially liquid phase to substantially gel phase,substantially liquid phase to substantially solid phase, or the like.Non-limiting examples of phase changes of a droplet that is fluidizedinclude substantially solid phase to substantially liquid phase,substantially gel phase to substantially liquid phase, or the like.

As mentioned above, in some embodiments, a species may be internallycontained in a rigidified droplet, such as a microfluidic droplet. Thespecies may be contained within the droplet during the initial formationof the droplet, or after the droplet has formed. In one set ofembodiments, a droplet is first rigidified, and then exposed to a fluidcontaining the species. The species may enter the droplet, for example,via diffusion. The species may then be contained within the droplet, forinstance, by chemically reacting the species to the droplet, or byreplacing the fluid with a second fluid that does not allow the speciesto substantially leave the droplet (e.g., due to a difference inhydrophobicity or hydrophilicity).

The following discussion gives non-limiting examples of methods to addat least one species to a rigidified droplet. In other cases, thespecies may be added to the droplet by injecting the species into thedroplet. In yet another case, the species may be a part of the firstfluid, such that the species is present in the droplet (e.g., in thefluid) before the droplet is formed, rigidified and/or fluidized. Othermethods and techniques for internally containing a species in arigidified droplet will be known to those of ordinary skill in the art.In some embodiments, exposing a droplet to a fluid comprising aplurality of species may cause at least one species to be containedinternally in the droplet. This may be achieved, in some cases, when thefirst fluid contained in the droplet and second fluid substantiallysurrounding the droplet are substantially miscible, and the second fluidcomprises a plurality of a type of species. At least a portion of thefluid contained in the droplet and the fluid surrounding the droplet mayexchange and cause at least one species to be contained internallywithin the first droplet.

Without wishing to be bound by theory, the substantial miscibility ofthe first fluid contained in the droplets and the second fluidsurrounding the droplet may allow for this exchange in some cases. Forexample, when second fluid comprising at least one species is exchangedwith at least some of the first fluid contained in the droplet, speciescomprised within the second fluid may also be transferred and containedinternally within the droplet. In embodiments where the first fluidcomprised within the rigidified droplet and the fluid surrounding thedroplet are substantially immiscible, the first fluid contained withinthe droplet may not substantially exchange with the fluid substantiallysurrounding the droplet.

A non-limiting illustration of this exchange is depicted in FIG. 2. InFIG. 2A, a rigidified droplet 50 containing a first fluid issubstantially surrounded by second fluid 52 comprising a plurality ofspecies 54. First fluid 50 and second fluid 52 are substantiallymiscible in this example, and fluid exchange between the rigidifieddroplet and the second fluid may occur, as indicated by arrow 56. Atleast one of plurality of species 58 may be transferred into rigidifieddroplet 50 during the fluid exchange and the fluid surrounding thedroplet 60 will now comprise the second fluid and at least some of thefirst fluid that was contained in the rigidified droplet 50. As anotherexample, as shown in FIG. 2B, a rigidified droplet 62 containing a firstfluid is substantially surrounded by second fluid 69 comprising aplurality of species 66. First fluid 60 and second fluid 62 aresubstantially immiscible and fluid exchange does not occur between therigidified droplet and the second fluid, as indicated the cross througharrow 64.

A rigidified droplet comprising a first fluid and surrounded by a secondfluid, may be exposed to a third fluid (e.g., comprising a plurality ofa type of species) using any technique known to those of ordinary skillin the art. For example, the rigidified droplet may be removed from thesecond fluid and transferred to the third fluid, as depicted in FIG. 6A.In this figure, rigidified droplet 200 is substantially surrounded bysecond fluid 202. The rigidified droplet may be removed (e.g., removedusing a pipette, tweezers, a spoon, vacuum, etc.) from second fluid 202,as indicated by arrow 201. Rigidified droplet 200 may then be added tothird fluid 204, as indicated by arrow 203.

As another example, a third fluid may be added to the second fluid, andin instances where the second fluid and the third fluid aresubstantially immiscible, the third fluid and the second fluid may formone or more layers. The rigidified droplet may then be substantiallysurrounded the third fluid. An example of this process for replacing thesecond fluid with a third fluid is depicted in FIG. 6B. Rigidifieddroplet 206 is substantially surrounded by second fluid 208. Third fluid210 may be added to second fluid 208. If second fluid 208 and thirdfluid 210 are substantially immiscible, third fluid 210 will form alayer above (as indicated by arrow 205) or below second fluid 208 (notshown). Rigidified droplet 206 may then be substantially surrounded byfluid 210, as indicated by arrow 207. The second and/or third fluid maybe agitated (e.g., stirred, centrifuged, etc.) to aid in the exposure ofthe rigidified droplet to the third fluid.

In yet another example, the second fluid may be removed fromsubstantially surrounding the droplets (e.g., evaporation of the secondfluid, aspiration or decanting of the second fluid, etc.), as depictedin FIG. 6C. Rigidified droplet 212 is substantially surrounded by secondfluid 214 in this example. Second fluid 214 is then removed (e.g.,draining of the second fluid) from substantially surrounding therigidified droplet, as indicated by arrow 213. Third fluid 218 can thenbe provided and substantially surround rigidified droplet 212, asindicated by arrow 215. In some cases, the rigidified droplets may moveinto the third fluid, e.g., via gravity or differences in buoyancy.

In some embodiment, after a species has been contained internally in arigidified droplet, the species may be immobilized relative to therigidified droplet. Those of ordinary skill in the art will be aware ofmethods to immobilize a species with respect to a composition (e.g.,gel, polymer) of a rigidified droplet. The species may be immobilizedwith respect to the composition of the droplet either directly (e.g.,formation of a bond, such as a covalent bond) or indirectly (e.g., usinga crosslinking molecule). In some instances, application of light orheat to a rigidified droplet internally containing a species may causethe species to become immobilized relative to the rigidified droplet. Asanother example, the species may be immobilized by exposure to aimmobilizing agent (e.g., a chemical compound).

In some cases, more than one species of a single type will be containedinternally in a rigidified droplet. For example, at least about 2species, at least about 3 species, at least about 5 species, at leastabout 10 species, at least about 20 species, at least about 50 species,at least about 100 species, and the like, may be added to a rigidifieddroplet. In some instances, more than one type of species will becontained in the rigidified droplet. That is, at least one of a firsttype of species and at least one of a second type of species may beadded to a rigidified droplet. This may be accomplished, for example,using the above techniques, where the fluid substantially surroundingthe droplet (which may be substantially miscible with the fluidcontained within the droplet) comprises a plurality of the first type ofspecies and a plurality of the second type of species. The total numberof a species in each of a plurality of droplets may or may notnecessarily be equal. For example, a first droplet in a plurality ofdroplets may comprise only one species of a single type and seconddroplet in the plurality of droplets may comprise more than one speciesof a single type.

In some cases, a rigidified droplet may comprise at least about 2 types,at least about 3 types, at least about 4 types, at least about 5 types,at least about 6 types, at least about 8 types, at least about 10 types,at least about 15 types, at least about 20 types, or the like, ofspecies. The total number of species of each type contained within thedroplet may or may not necessarily be equal. For instance, in somecases, when two types of species are contained within a droplet, theremay be approximately an equal number of the first type of species andthe second type of species contained within the rigidified droplet. Inother cases, the first type of species may be present in a greater orlesser amount than the second type of species, for example, the ratio ofone species to another species may be about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:10, about 1:20, about 1:100, and the like.The number of species of each type of species in each of a plurality ofdroplets in a group may or may not be equal. For example, a firstdroplet of a group may comprise one species of a first type and onespecies of a second type where a second droplet of the group may containmore than one species of the first type and one or more species of thesecond type.

The species may or may not be substantially soluble in the fluid containin the droplet and/or the fluid substantially surrounding the droplet.For example, in some cases, a species may be substantially soluble in asecond fluid substantially surrounding a droplet and in a first fluidcontained within a droplet. In other cases, the species is substantiallysoluble in the second fluid and substantially insoluble in the firstfluid, such that the species may precipitate when contained in thedroplet. In yet other cases, the species is substantially insoluble inboth the second fluid and the first fluid, and is suspended in thefluids.

The methods for forming a plurality of species-containing dropletsand/or a suspension of rigidified species-containing droplets may be, insome embodiments of the present invention, applied to creating librariesof droplets containing various species. For example, in someembodiments, a plurality of rigidified droplets may be separated into atleast a first group and at least a second group of rigidified droplets,where a distinguishable type of species may be contained internally inat least some of each group of droplets. At least one droplet from eachgroup may be combined to form a plurality of droplets, each comprisingat least one distinguishable species (e.g., a library of droplets) Thatis, at least one first species and one least second species may be addedto at least a portion of the first group and the second group ofrigidified droplets, respectively, and at least one droplet from eachgroup may be combined to form a suspension of rigidified droplets (e.g.,a library). Addition of a species to a droplet may be accomplished usingany of the techniques discussed herein. In a particular embodiment, afirst group of droplets and a second group of droplets are exposed to afirst fluid and a second fluid comprising a plurality of a first speciesand a second species, respectively. The plurality of rigidified dropletsmay be separated into at least about 2 groups, at least about 4 groups,at least about 10 groups, at least about 30 groups, at least about 50groups, at least about 64 groups, at least about 128 groups, at leastabout 1024 groups, at least about 4096 groups, at least about 10,000groups, and the like. The number of groups the plurality of droplets isseparated into may be selected such that the number is approximatelyequal to the total number of types of distinguishable species to beadded to the droplets.

In some embodiments, a suspension may be formed that comprises at leasta portion of each group of rigidified droplets. For example, a portionof a first group and of a second group of rigidified droplets may besuspended in a fluid. The first and second groups of rigidified dropletsmay be combined into a common suspension of the first and second groupsof droplets using any known technique. For example, a first fluidcomprising the first group of rigidified droplets and the second fluidcomprising the second group of rigidified droplets may be combine toform a third fluid (e.g., comprising the first and the second fluids)comprising the first group and the second group of rigidified droplets.In some cases, only a portion of the first fluid comprising a portion ofthe first group of rigidified droplets will be combined with a portionof the second fluid comprising a portion of the second group ofrigidified droplets. The first fluid and the second fluid may or may notcomprise substantially the same make-up. In certain aspects, the firstfluid and the second fluid will be miscible. In some cases, thesuspension of rigidified droplets may be exposed to a third fluid usingthe techniques discussed here (e.g., see FIG. 6).

A suspension of rigidified droplets (e.g., a library of rigidifieddroplets) may be stored for any length of time. In some cases, thesuspension of rigidified droplets may be stored such that the speciescontained within each droplet does not substantially change. That is,the species contained within each droplet do not diffuse from thedroplet and/or new species are not contained internally in the droplet.Without wishing to be bound by theory, this aspect of the invention maybe important during the formation of a library comprising labels. Forexample, if a label contained in a first droplet is able to exchangewith a label contained in a second droplet, where the labels are eachassociated with a particular species in a library, the labels would berendered useless in determining the species of the library contained inthe droplet if exchange is possible. For example, at least about 100%,at least about 99.5%, at least about 99%, at least about 98%, at leastabout 97%, at least about 96%, at least about 95%, at least about 90%,at least about 85%, at least about 80%, and the like, of the pluralityof rigidified droplets contained in the suspension may contain the samespecies after storage as they did prior to storage. The suspension ofthe droplets may be stored without substantial change for at least about1 day, at least about 2 days, at least about 5 days, at least about 10days, at least about 1 month, at least about six months, at least about1 year, at least about 2 years, at least about 5 years, and the like.

Prior to forming the suspension of droplets, in some embodiments, agroup of droplets containing at least one species may be exposed to afluid which does not comprise any species. Without wishing to be boundby theory, exposure of the group of rigidified droplets containing aspecies to a fluid which is substantially immiscible with the dropletsmay prevent the species contained within the droplet from beingtransferred to the fluid surrounding the droplets through fluid exchange(e.g., because the fluids are not miscible, the exchange of the twofluids will be little or none). For example, a group of rigidifieddroplets containing at least one species and a first fluid may beexposed to a second fluid that does not comprise a plurality of species,where the first fluid and the second fluid are substantially immiscible.The immiscibility of the first fluid with the second fluid may not allowfor the species contained in the droplet to diffuse into the secondfluid. In other instances, when the species are immobilized relative tothe droplet, the group of rigidified droplets may be exposed to a fluidwhich is substantially immiscible or substantially miscible with thefluid contained in the droplet since the species are immobilizedrelative to the droplet, the species may not be able to diffuse into thefluid substantially surrounding the droplet.

In some embodiments, a plurality of microfluidic droplets may be formed,where each of the microfluidic droplets comprises at least onespecies-containing rigidified or fluidized droplet (e.g., from a libraryof droplets) and in some cases, at least one target analyte molecule.Within the microfluidic droplet, the target analyte molecule mayinteract with a species for the species-containing droplets, and in somecases, the interaction may be determined. In instances where thespecies-containing droplets are fluidized, the fluidized droplet and theanalyte droplet may be fused to form a single droplet containing thefluids from both droplets. This may occur if the fluid within theanalyte droplet and the fluid in the fluidized droplet are substantiallymiscible. In instances where the species-containing droplet isrigidified, the rigidified droplet may be contained within the analytedroplet. A plurality of rigidified droplets may be fluidized using themethods and techniques discussed herein.

In some cases, a plurality of microfluidic droplets (e.g., fuseddroplets) may be formed by fusing at least a portion of a plurality ofspecies-containing droplets (e.g., a library of droplets) with aplurality of analyte droplets containing analyte molecules. An “analytedroplet” is a droplet which contains at least one fluid and at least onetarget analyte molecule. The plurality of species-containing dropletsmay be rigid of fluidic. At least a portion of the species-containingdroplets (either rigidified or fluidized) may be fused with a pluralityof analyte droplets using microfluidic techniques, such as thosedescribed in U.S. patent application Ser. No. 11/024,228, filed Dec. 28,2004, entitled “Method and Apparatus for Fluid Dispersion,” by Stone, etal., published as U.S. Patent Application Publication No. 2005/0172476on Aug. 11, 2005; U.S. patent application Ser. No. 11/246,911, filedOct. 7, 2005, entitled “Formation and Control of Fluidic Species,” byLink, et al., published as U.S. Patent Application Publication No.2006/0163385 on Jul. 27, 2006; U.S. patent application Ser. No.11/885,306, filed Aug. 29, 2007, entitled “Method and Apparatus forForming Multiple Emulsions,” by Weitz, et al.; or U.S. patentapplication Ser. No. 11/360,845, filed Feb. 23, 2006, entitled“Electronic Control of Fluidic Species,” by Link, et al., published asU.S. Patent Application Publication No. 2007/0003442 on Jan. 4, 2007,each incorporated herein by reference.

In some cases, a plurality of fluidized species-containing droplets arefused with a plurality of analyte droplets. The fluid contained withinthe analyte droplet and the fluid contained within the suspensions ofdroplets, in most cases, may be substantially miscible. This may, incases where the plurality of species-containing droplets are fluidized,allow for essentially complete fusion of a fluidized droplet with theanalyte droplet, such that the fused microfluidic droplet formedcontains the species from the fluidized droplet and the target analytemolecule. For example, as shown in FIG. 3A, fluidized droplet 100 fromthe suspension containing species 102 and analyte droplet 104 containinga target analyte molecule 106 are fused, as indicated by arrow 107, toform fused fluidic droplet 108 comprising species 102 and target analytemolecule 106.

In other cases, the suspension of rigidified species-containing dropletsare fused with a plurality of analyte droplets to form a plurality offused microfluidic droplets. In some aspects of this embodiment, thefluid contained in the rigidified droplets may be substantially misciblewith the fluid contained in the analyte droplet and may allow for thespecies contained within the rigidified droplet to diffuse from therigidified droplet into the fluid of the fused microfluidic droplet, orfor the target analyte molecule to be contained internally in therigidified droplet, thus allowing the target analyte molecule and thespecies to interact. As a non-limiting example, as shown in FIG. 3B,species-containing rigidified droplet 110 containing species 112 andanalyte droplet 114 containing target analyte molecule 116 are fused, asindicated by arrow 107, such that the fused microfluidic droplet formed118 contains target analyte molecule 116 and rigidified droplet 120containing species 112. The miscibility of the fluid contained in fusedmicrofluidic droplet 118 and rigidified droplet 120 may allow for theexchange of fluid, as indicated by arrow 122. This may allow for species116 to diffuse into the fluid of the fused droplet 124 from rigidifieddroplet 126, as indicated by arrow 121. In other aspects, a least aportion of the species contained in the rigidified droplet may remaincontained internally in the rigidified droplet while interacting withthe target analyte molecule.

In yet another embodiment, a plurality of species-containing rigidifieddroplets in which the species have been immobilized relative to arigidified droplet may be fused with a plurality of analyte droplets.The interaction between the target analyte molecule and the speciesimmobilized relative to a rigidified droplet may be determined. In suchinstances, the target analyte molecule may be substantially soluble inthe fluid substantially surrounding the plurality of rigidifieddroplets. In addition, the fluid comprised in the rigidified dropletsmay be substantially miscible with the fluid substantially surroundingthe droplets. Without wishing to be bound by theory, this propertyshould allow for the target analyte molecule to diffuse into therigidified droplet, thereby interacting with the species immobilized inthe rigidified droplet.

In still yet another embodiment, a plurality of microfluidic dropletsmay be formed using microfluidic techniques from a solution comprising afluid, a suspension of rigidified species-containing droplets, and aplurality of target analyte molecules. For example, the plurality oftarget analyte molecules may be provided to the fluid substantiallysurrounding the plurality of rigidified droplets. Using microfluidictechniques, the fluid may be dispersed into droplets, where thedispersed droplets may comprise a target analyte molecule and/or arigidified droplet. This non-limiting example is depicted in FIG. 3C.Fluid 128 is provided comprising a plurality of target analyte molecules134 and a plurality of rigidified droplets 130 internally containingspecies 132. The continuous stream of fluid 128 may flow intomicrofluidic channel 138 comprising second fluid 136 to form a pluralityof microfluidic droplets 140, a technique that will be well known tothose commonly skilled in the art.

It should be understood when using the various embodiments discussedabove, not every microfluidic droplet formed will comprise a rigidifiedor fluidized droplet and a target analyte molecule. Some droplets formedmay contain neither a rigidified or fluidized droplet nor a targetanalyte molecule, some droplets formed may contain only one of the two,and some droplets formed may contain both. This by no means limits theapplications of the droplets formed. Additional methods for forming aplurality and/or suspension of rigidified droplets are now described.

In one embodiment of the present invention, a method for forming aplurality of species-containing rigidified droplets comprising firstproviding a plurality of droplets, each of the plurality of dropletscomprising a first fluid and being substantially surrounded by a secondfluid, where the first fluid and the second fluid are substantiallyimmiscible. The plurality of droplets may undergo a phase change to forma plurality of rigidified droplets (e.g., gel droplets). The pluralityof rigidified droplets may be exposed to a third fluid, which may, insome cases, be substantially miscible with the first fluid contained inthe rigidified droplets. At least one first species may be addedinternally to at least some of the rigidified droplets (e.g., bydiffusion of a fluid containing the species into the droplet).

A non-limiting example of the above method is depicted in FIG. 4. Aplurality of droplets 150 comprising a first fluid are substantiallysurrounded by a second fluid 152, where the first fluid and the secondfluid are substantially immiscible. The plurality of droplets undergo aphase change, as indicated by arrow 151, to form a plurality ofrigidified droplets 154, which are substantially surrounded by secondfluid 152. The plurality of rigidified droplets 154 are exposed to athird fluid 156, where the first fluid comprised in the plurality ofrigidified droplets 154 is substantially miscible with third fluid 156,as indicated by arrow 155. At least one first species 158 is added toeach rigidified droplet 160, as indicated by arrow 157.

As another embodiment, a method to form a plurality ofspecies-containing droplets may comprise the following steps. First, aplurality of groups of rigidified droplets may be provided. Each ofgroups of rigidified droplets may have substantially the samecomposition as the other groups of rigidified droplets, where each groupof droplet contains the same first fluid and at least onedistinguishable species with respect to the other groups of rigidifieddroplets. A suspension may be formed which contains at least onerigidified droplet from each of the groups of rigidified droplets. Atleast some of the suspension of droplets, in some cases, may be exposedto a second fluid, where the second fluid is substantially immisciblewith the first fluid. Exposure of the rigidified droplets to a secondfluid which is substantially immiscible with the first fluid containedin the droplets may allow for at least some of the droplets to befluidized such that the droplets do not coalesce with the fluidsurrounding the droplets.

A non-limiting example is illustrated in FIG. 5. In this example, threegroups of rigidified droplets (172, 176, 178) are provided, each dropletcomprising a first fluid and at least one type of species (172, 178,184, respectively), where each of the types of species aredistinguishable with respect to the others species. Each of the groupsof rigidified droplets are substantially surrounded by a second fluid(174, 180, 186), where the first fluids and the second fluids aresubstantially immiscible. Each of the groups of droplets havesubstantially the same make-up (e.g., each of the droplets consistsessentially of the same material). The second fluid surrounding each ofthe groups of rigidified droplets may be the same or different fluid. Asuspension 186 is formed which comprises at least one rigidified dropletfrom each of the three groups of droplets (171, 177, 183) suspended influid 184, as indicated by arrow 173. The suspension of droplets 186 maybe exposed to a third fluid 188, as indicated by arrow 175. Thesuspension of droplets may then be fluidized, as indicated by arrow 179,to form a plurality of fluidized droplets 196 comprising a fluidizeddroplet from each group (190, 192, 194), each comprising adistinguishable species (172, 178, 184).

In yet another embodiment, the present invention provides a method forforming a suspension of species-containing droplets comprising at leastone droplets from each of a first group of rigidified droplets and asecond group of rigidified droplets, where the first group of rigidifieddroplets contains a first species which is distinguishable from thesecond species contained in the second group of rigidified droplets. Themethod may first provide a first plurality of rigidified dropletscontaining a first fluid, where each rigidified droplet is substantiallysurrounded by a second fluid which is substantially immiscible in thefirst fluid. The first plurality of rigidified droplets may be exposedto a third fluid, where the third fluid is substantially miscible in thefirst fluid. At least one first species may be added internally to atleast some of the first plurality of rigidified droplets to form a firstgroup of species-containing droplet. The above steps may be repeatedwith a second plurality of rigidified droplets and a second species toform a second group of species-containing droplets. A suspension may bethen formed which contains at least some droplets of the first andsecond groups of species-containing droplets.

An example of the above is depicted in FIG. 8. A first plurality ofrigidified droplets 240 comprising a first fluid is provided, where eachof the rigidified droplets is substantially surrounded by a second fluid242. The first plurality of rigidified droplets 240 are exposed to athird fluid 244, as indicated by arrow 241. A least one first species248 is added to at least some of the first plurality of rigidifieddroplets to form a first group of species-containing droplets 248, asindicated by arrow 243. These series of steps 248 are performed with asecond plurality of rigidified droplets 250, thereby forming a secondplurality of species-containing droplets 254 comprising a second species252, where first species 248 and second species 252 are distinguishablewith respect to each other. A suspension 256 is then formed comprisingat least one first species-containing droplet 246 and at least onesecond-species containing droplet 252, as indicated by arrow 245.

As mentioned above, the methods for forming a plurality ofspecies-containing droplets and/or a suspension of rigidifiedspecies-containing droplets may be, in some embodiments of the presentinvention, applied to creating libraries of droplets containing variousspecies. A library may contain droplets that are all substantially thesame size and have substantially the same composition, but differ in thespecies contained within the droplets. For instance, a first member of alibrary may be a collection of droplets containing a first species, anda second member of a library may be a collection of droplets containinga second species, the first species at a different concentration, afirst species and a second species, or the like. Such libraries may beuseful, for example, for nucleic acid sequencing applications, screeningassays, or the like.

In some cases, the library includes compositions comprising a containercomprising a plurality of mutually distinguishable rigidified droplets,each having substantially the same composition but containing adistinguishable species with respect to the other rigidified droplets.In some cases, the composition is produced using the methods discussedherein. The composition may comprise at least about 5, at least about 8,at least about 10, at least about 20, at least about 50, at least about64, at least about 100, at least about 128, at least about 200, at leastabout 500, at least about 1000, at least about 4096, at least about10,000, at least about 50,000, and the like, mutually distinguishablespecies. The substantially same composition of the rigidified dropletsmay comprise a polymer and/or gel.

The composition and methods of the present invention may be useful forthe sequencing of a target nucleic acid. For example, the target analytemolecule may be a nucleic acid and the species may be selected from alibrary of nucleic acid probes, such that the sequence of the nucleicacid may be determined. Other target analyte molecules that can bestudied include, for example, biochemical species such as nucleic acidssuch as siRNA, RNAi and DNA, proteins, peptides, or enzymes. It shouldbe understood, however, that while the discussion herein primarilyfocuses on the use of droplets of the present invention for sequencing anucleic acid, this is by way of example only, and the droplets have manyother uses, such as techniques relating to fields such as food andbeverages, health and beauty aids, paints and coatings, and drugs anddrug delivery. For example, in some instances, a target analyte moleculemay be a cell, and the interaction of the cell with a variety of species(e.g., drugs, hormones, etc.) may be determined. Species that can beincorporated within droplets of the invention include, but are notlimited to, nucleic acid probes, nanoparticles, quantum dots,fragrances, proteins, indicators, dyes, fluorescent species, chemicals,or the like. A droplet or emulsion can also serve as a reaction vesselin certain cases, such as for controlling chemical reactions, or for invitro transcription and translation, e.g., for directed evolutiontechnology. In addition, droplets of the present invention may compriseadditional reaction components, for example, catalysts, enzymes,inhibitors, and the like.

In some embodiments, the target analyte molecule may be a target nucleicacid to be sequenced, where the target nucleic acid may be any suitablenucleic acid. For example, the target nucleic acid may be a nucleic acidthat encodes a biological entity, such as a protein, an enzyme, anantibody, a receptor, a ribozyme, a ribosome, or the like, and/or aportion thereof. As another example, the target nucleic acid may be aregulatory sequence or a non-coding sequence, for instance, a smallinterfering RNA, a microRNA, a small hairpin RNA, or the like. Thetarget nucleic acid can be any number of nucleotides in length, forexample, on the order of 25, 50, 60, 64, 70, 80, 90, 100, 200, 400, 800,1600, 3200, 6400, or even more nucleotides in length. Non-limitingexamples of target nucleic acids include ribonucleic acid (RNA),deoxyribonucleic acid (DNA), or mixtures or copolymers thereof, whichmay be isolated from natural sources, recombinantly produced,artificially synthesized, etc. The nucleic acid may contain residuessuch as adenosine or “A,” thymidine or “T,” guanosine or “G,” cytidineor “C,” or uridine or “U,” or other residues, such as the universalresidues. The nucleic acid can be double-stranded or single stranded tofacilitate hybridization. Moreover, the nucleic acid can be obtainedfrom virtually any source. For instance, the nucleic acid may beisolated from a cell or a virus, synthesized using traditional chemicalsynthesis, synthesized using polymerase chain reaction (PCR) technology,or the like.

The target nucleic acid contained within the droplet may be exposed to anucleic acid probe and/or one or more identification elements. Forinstance, as previously discussed, an analyte droplet comprising atleast one target analyte molecule (e.g., a target nucleic acid) may befused with a species-containing droplet (e.g., a nucleic acid probe) andin most cases, at least one identification element for determining thenucleic acid probe. The fluidic droplets may be used for sequencing anucleic acid using techniques such as those disclosed in U.S. PatentApplication Ser. No. 61/008,862, filed Dec. 21, 2007, entitled “Systemsand Methods for Nucleic Acid Sequencing,” by Weitz, et al, hereinincorporated by reference.

The suspension of droplets (either fluidized or rigidified) may comprisea plurality of group of droplets, where each group of droplet internallycontains at least one nucleic acid probe. Nucleic acid probes aregenerally used, in certain embodiments, to determine certain sequenceswithin the target nucleic acid. Often, short portions of the targetnucleic acid can be associated with the nucleic acid probe, forinstance, a sequence of less than 20 residues, less than 15 residues,less than 10 residues, less than 9 residues, less than 8 residues, lessthan 7 residues, less than 6 residues, less than 5 residues, less than 4residues, etc. The residues are typically contiguous within the targetnucleic acid probe although, in some cases, some of the residues withinthe target nucleic acid are not necessarily contiguous. In someembodiments, a nucleic acid probe may contain a relatively shortsequence of nucleic acid residues that is able to recognize at least aportion of the target nucleic acid, and often has a similar length asthe recognized portion of the target nucleic acid. For instance, thenucleic acid probe may have a sequence having length of less than 20nucleotides or less than 10 nucleotides in some cases, or a length suchas those described above. In one case, the length of the nucleic acidprobe sequence may be four residues (e.g., FIG. 7A). In another case,the length may be five residues (e.g., FIG. 7B). In yet another case,the length may be six residues (e.g., FIG. 7C). The nucleic acid probesequences within the nucleic acid probe may be contiguous, or thesequence may be non-contiguous. For instance, there may be universalresidues or gaps present. In some instances, the nucleic acid probe maybe labeled in some manner, such as with a signaling entity, forinstance, a radioisotope or with a fluorescence tag (e.g., FIG. 7D).Various signaling entities and other examples of nucleic acid probeswill be discussed in more detail below.

The nucleic acid probe may be selected such that at least some of theprobes will contain sequences complementary or substantiallycomplementary to the target nucleic acid sequence. For instance, in oneembodiment, the nucleic acid probe sequences are selected such thatevery permutation of nucleic acid residues of a certain size or number(or range of sizes or numbers) is represented, thereby ensuring that atleast one of those nucleic acid probe sequences is substantiallycomplementary to the target nucleic acid. As used herein, a firstsequence that is “substantially complementary” to a second sequence isone which at least about 75% of the first and second sequences arecomplementary (e.g., through Watson-Crick complementarity pairing)and/or the sequences have a maximum of 1 or 2 base mismatches. In someembodiments, the two sequences may be at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 97%, atleast about 98%, at least about 99% or at least about 100%complementary.

In some embodiments, a plurality of distinguishable or non-identicalnucleic acid probes is used, for example, nucleic acid probes having oneor more differences in the sequence of residues contained within thenucleic acid probes. For instance, a plurality of fluidized orrigidified droplets may be used, and the droplets may each contain aspecific nucleic acid probe sequence. The droplets may be prepared suchthat each droplet contains only one nucleic acid probe sequence(although multiple copies of the nucleic acid-probe may be present). Inaddition, in some cases, different droplets may independently containthe same or different nucleic acid probe sequence (e.g., such that thereis some redundancy so that not each droplet in a given population orcollection of droplets is necessarily unique).

In some cases, the nucleic acid probe may be labeled, e.g., with asignaling entity. The signaling entity may be determined in some fashionusing a detection method, such as those discussed herein. The signalingentity may be included within the nucleic acid probe at any suitablelocation, for example, at a 5′ terminal site of the nucleic acidsequence of the nucleic acid probe, a 3′ terminal site, or at aninternal site within the nucleic acid probe. In some cases, thesignaling entity may be chosen such that it produces a different signal(or does not produce a signal) when the nucleic acid probe is associatedwith a target nucleic acid compared to when the nucleic acid probe isnot associated with the target nucleic acid. The signaling entity mayinclude, but is not limited to, a fluorescent dye, a chemiluminescententity, a radioactive label, an isotope such as a non-radioactiveisotope or an isotope detectable by mass spectrometry (e.g., anelectrophore mass label (EML)), a ligand which can serve as a specificbinding partner to a labeled antibody, an enzyme, an antibody which canserve as a specific binding partner for a labeled ligand, an antigen, agroup having a specific reactivity, and/or an electrochemicallydetectable moieties. Non-limiting examples of fluorescent signalingentities include fluorescein, rhodamine, or hexachlorofluorescein; thoseof ordinary skill in the art will be aware of other fluorescent entitiesthat are readily commercially available. Yet other examples of signalingentities are discussed in detail herein.

For instance, in one embodiment, a nucleic acid probe can include asequence of nucleic acid residues, a signaling entity, and a quencher oran enhancer (e.g., as is shown in FIG. 7E, with the signaling probelabeled S and a quencher labeled Q). The signaling entity may be, e.g.,a fluorescent entity, and may be located anywhere in the nucleic acidprobe, for instance, covalently attached to the 5′ end of the nucleicacid sequence. Non-limiting examples of fluorescent entities potentiallysuitable for use in the nucleic acid probe in various embodimentsinclude 6-carboxyfluorescein and tetrachlorofluorescin. The quencher orenhancer may be any entity able to affect the signaling entity in somefashion, e.g., by respectively inhibiting or facilitating determinationof the signaling entity. For instance, the proximity of a fluorescentsignaling entity and a quencher within a nucleic acid probe may be suchthat the quencher is able to partially or completely inhibit fluoresenceof the signaling entity, while an enhancer may be able to enhance thefluorescence of a fluorescent signaling entity when the enhancer ispositioned proximate the signaling entity. The quencher or enhancer mayalso be located anywhere in the nucleic acid probe, for example,attached to the 3′ end of the nucleic acid sequence. Non-limitingexamples of quenchers include tetramethylrhodamine anddihydrocyclopyrroloindole tripeptide.

As a non-limiting example, a quencher (or similarly, an enhancer) can beused within a signaling entity in a nucleic acid probe as follows. Anucleic acid probe associated with a target nucleic acid may be removedor dissociated from the target nucleic acid by the action of certainenzymes or other species, for instance, polymerases such as Taqpolymerases. For instance, in some cases, a polymerase may causedegradation of the nucleic acid sequence within the nucleic acid probeto occur, which may cause release of the signaling entity and/or thequencher or enhancer and hence, the quencher or enhancer may no longerbe proximate to or at least substantially affect the signaling entity.Thus, degradation of the nucleic acid probe can be determined bydetermining a change in the signaling entity. In contrast, in systemswhere the nucleic acid probe does not sufficiently associate with thetarget nucleic acid (e.g., if no sufficiently complementary sequencesare present), no degradation of the nucleic acid probe would occurthrough action of the polymerase or other species (e.g., any associationthat exists between the target nucleic acid and the nucleic acid probeis too transient or short for enzymatic action to occur), and thus, nosignificant change in the signal of the signaling entity could bedetermined. Accordingly, in one embodiment, a polymerase such as Taqpolymerase may be provided to a fluidic droplet comprising a nucleicacid probe and a target nucleic acid. The polymerase may be provided tothe fluidic droplet using any suitable technique, as discussed herein.

In some cases, a nucleic acid probe may comprise at least one lockednucleic acid (LNA) residue (see, e.g., FIG. 7F). A locked nucleic acidresidue is a nucleic acid analog that has a chemical shape similar to anaturally occurring nucleic acid residue (e.g., being able to form 2 or3 hydrogen bonds with a complementary residue), but is not free torotate in as many dimensions as a naturally occurring nucleic acidresidue. For instance, in some cases, a locked nucleic acid residue maycontain a 2′-O, 4′-C methylene bridge, where the methylene bridge“locks” the ribose in the 3′-endo structural conformation, which isoften found in the certain form of DNA or RNA. The locked riboseconformation may enhance residue stacking and/or backbonepre-organization. This can significantly increase the thermal stability(melting temperature) of the nucleic acid sequence in some cases. Anucleic acid probe containing one or more locked nucleic acid residuesmay be useful in certain embodiments because the locked nucleic acidresidue may exhibit increased affinity for association with the targetnucleic acid, e.g., due to the restrictions on its ability to internallyrotate.

In certain embodiments, the nucleic acid probe may contain a universalresidue, which may be able to engage in a residue-pairing relationshipwith more than one natural nucleotide, and in some cases, with all ofthe natural nucleotides. Exemplary universal residues include5-nitroindole and 3-nitropyrrole, although other universal residuesuseful for the systems and methods described herein will be known tothose of skill in the art. As discussed below, a nucleic acid probecontaining one or more universal bases may be useful in certainembodiments.

The nucleic acid probes may be synthesized using any suitable technique,e.g., solid phase phosphoramidite triester methods. In some cases, aplurality of nucleic acid probes is synthesized, forming a library ofsuch probes. The library may include a plurality of sequences, forexample, organized in a plurality of droplets. In some (but not all)embodiments, the library may contain sequences that have roughly thesame number of residues, for example, around 4 residues, around 5residues, around 6 residues, around 7 residues, etc. The library ofnucleic acid probes may be prepared using any suitable technique, andmay be produced using manual techniques or automated, e.g., using arobotic apparatus.

In one embodiment, the library may comprise every possible sequence fora set of nucleic acid sequences having a certain length or lengths. Inanother embodiment, the library may comprise at least about 30%, atleast about 50%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 100% of all possiblesequences having a certain length or lengths. Some techniques forpreparing a library are discussed below.

The library of nucleic acid probes (or any other species) may beinternally contained within droplets of the present invention using anysuitable technique, as discussed herein. For example, a plurality ofrigidified droplets may be produced microfluidically, such that theplurality of rigidified droplets have substantially the same compositionas all other droplets of the present invention. A library of nucleicacid probes may be provided (e.g., prepared on a microtiter plate usinga robotic apparatus), where each type of distinguishable nucleic acidprobe is kept separate from all other types nucleic acid probes. Aplurality of rigidified droplets may be separated into approximately thesame number of groups as there is types of nucleic acid probes, and eachgroup of rigidified droplets may be exposed to one type of nucleic acidprobe, such that at least one of each type of nucleic acid probe iscontained internally in a ridigified droplet, forming a plurality ofgroups of distinguishable droplets (e.g., each group containing adistinguishable nucleic acid probe). At least one of each type ofrigidified droplet containing interally a distinguishable nucleic acidprobe may be combined to form a suspension off rigidified droplets.

In many embodiments, at least one identification element may also becontained internally in each group of fluidized or rigidified droplets.An “identification element” as used herein, is a species that includes acomponent that can be determined in some fashion, e.g., theidentification element may be identified when contained within adroplet. The identification elements may be insoluble (e.g., suspended)or soluble within the droplet. Non-limiting examples includeidentification elements detectable by fluorescence, chemiluminescence,radioactivity, or the like. Specific examples include, but are notlimited to, particles containing dyes, quantum dots, or fluorescentparticles which, in some embodiments, may also have other speciesattached thereto, for instance, oligonucleotides such as those describedherein. In some cases, more than one identical identification elementmay be present within any given droplet.

In certain embodiments, more than one non-identical identificationelement may be used, e.g., within a droplet. For instance, a droplet maycontain at least two distinguishable identification elements, at leastthree distinguishable identification elements, at least fourdistinguishable identification elements, at least five distinguishableidentification elements, etc. Identification elements may bedistinguished using any suitable method, e.g., color, fluorescence,absorption, intensity, size, charge, radioactivity, mass, or the like.

In one set of embodiments, particles or microparticles (e.g., beads) maybe used as identification elements. The particles may have anydimension, and may be spherical or non-spherical. For instance, theparticles may have average diameters ranging from approximately 100 nmto 100 um in diameter in some cases. In certain embodiments, theparticles may have an average diameter of less than about 1 micrometer,less than about 300 nm, less than about 100 nm, less than about 30 nm,or less than about 10 nm. The average diameter, as used herein, is thearithmetic average of the diameters of the particles contained withinthe droplets. The diameters of a non-spherical particle is the diameterof a perfect mathematical sphere having the same volume as the particle.

In some embodiments, a plurality of identification elements may bechosen to identify droplets such that there are at least 3distinguishable identification elements, at least 4 distinguishableidentification elements, at least 6 distinguishable identificationelements, at least 8 distinguishable identification elements, at least 9distinguishable identification elements, at least about 10distinguishable identification elements, at least about 20distinguishable identification elements, at least about 30distinguishable identification elements, at least about 40distinguishable identification elements, at least about 50distinguishable identification elements, at least about 60distinguishable identification elements, at least about 70distinguishable identification elements, at least about 80distinguishable identification elements, at least about 90distinguishable identification elements, at least about 100distinguishable identification elements, etc. One non-limiting exampleof a plurality of distinguishable identification elements are theLuminex FlexMAP Microspheres beads commercially available from LuminexCorp. Beads or particles such as these may be distinguished, accordingto one embodiment, by the use of two or more dyes or other compoundsthat can be independently varied within each bead or particle.Therefore, a plurality of distinguishable beads may be used as aplurality of identification elements, according to certain embodiments.As another, specific non-limiting example, particles comprisingpolystyrene and one or more dyes may be used as identification elements.The dyes employed within the particles may include, for instance,squaric acid-based molecules or other fluorescent molecules that exhibitfluorescence, e.g., extending into near infrared and/or infrared region.In some cases, two or more dyes with concentrations that can beindependently controlled can be used within each particle.

The sequence of a target nucleic acid may be determined by determiningthe association (or non-association) of the target nucleic acid to oneof a plurality of distinguishable nucleic acid probes. The targetnucleic acid may be associated with the nucleic acid probe when theyform a relatively stable duplex by hydrogen bonding under experimentalconditions. Relatively stable hydrogen bonding may be formed due toWatson-Crick complementarity (e.g., A matches T, but not G or C; Gmatches C, but not A or T) and/or other effects such as GC wobble, orother associations caused by locked nucleic acids or universal bases, asdiscussed herein. Non-limiting examples of suitable methods fordetermining the sequence of a target nucleic acid include sequencing byhybridization techniques that are known to those of ordinary skill inthe art.

Sequencing by hybridization (SBH) is a method for examining the nucleicacid residue sequence in a target nucleic acid that has been previouslydescribed, for instance, in U.S. Pat. No. 5,202,231. In general, SBHuses a set of nucleic acid probes of defined sequence to probe forcomplementary sequences on a longer target strand of a target nucleicacid. The defined sequences which hybridize to the target can then bealigned using computer algorithms to construct the sequence of thetarget nucleic acid.

Thus, in one embodiment of the present invention, a target nucleic acidmay associate with a certain combination of nucleic acid probes, leadingto a characteristic “hybridization” pattern. Each positive association(or hybridization) event in a given sample provides a discrete piece ofinformation about the target nucleic acid. In some cases the targetnucleic acid may be sampled without determination of exactly where anyparticular nucleic acid probe associates with the target nucleic acid.Algorithms and software have been developed for target nucleic acidreconstruction, based on the hybridization pattern, and are known tothose skilled in the art. In other cases, however, analysis of ahybridization pattern, such as those described herein, may provide a“fingerprint” identification of the target nucleic acid sequence,without specifically determining the target nucleic acid sequenceitself. The pattern of hybridization may also be manually or computeranalyzed.

Another aspect of the present invention is generally directed to systemsand techniques for creating a suspension of rigidified or fluidizeddroplets, where the droplets contain distinguishable species and/oridentification elements. In some embodiments, a plurality ofdistinguishable identification elements may be used to identify aplurality of fluidic droplets, and in some cases, the distinguishableidentification elements are used to determine a nucleic acid sequence(e.g., of a nucleic acid probe) present within each droplet. Forinstance, in one embodiment, at least about 64, at least about 256, atleast about 1024, at least about 4096, or at least about 16,384 or morefluidic droplets may be prepared, each containing a nucleic acid probe(including multiple copies of the nucleic acid probe) and one or moreidentification elements that, in combination, identifies that nucleicacid probe and do not identify different nucleic acid probes. Thepresent invention provides, in one set of embodiments, systems, andmethods for preparing such collections of fluidized or rigidifieddroplets.

In one embodiment, a plurality of distinguishable identificationelements are used to identify a plurality of fluidic droplets or nucleicacid probes or other suitable samples. For instance, if fluorescentparticles are used, a set of distinguishable particles is firstdetermined, e.g., having at least 5 distinguishable particles, at leastabout 10 distinguishable particles, at least about 20 distinguishableparticles, at least about 30 distinguishable particles, at least about40 distinguishable particles, at least about 50 distinguishableparticles, at least about 75 distinguishable particles, or at leastabout 100 or more distinguishable particles. A non-limiting example ofsuch a set is available from Luminex. The distinguishable identificationelements may be divided into a plurality of groups (e.g., 2, 3, 4, 5, 6,7, or more), where each group contains at least two members of the setof distinguishable identification elements.

A sample may then be associated with one member chosen from each of thegroups of distinguishable identification elements. For instance, a firstsample may be identified by the combination of a first element chosenfrom a first group, a first element chosen from a second group, and afirst element chosen from a third group, as each of these elements isdistinguishable from each other; a second sample may be identified bythe combination of a first element chosen from the first group, a firstelement chosen from the second group, but a second element chosen fromthe third group. The number of unique combinations, in this example, issimply the product of the number of members of each of the groups; alarge number of distinguishable sets of identification elements can thusbe prepared. Thus, for instance, by defining at least six identificationelements, where the identification elements are arranged into at leastthree groups with each group having at least two identificationelements, at least eight different samples can be determined byassociating each of the at least eight samples with at least three ofthe identification elements, where each identification elementassociated with each sample is chosen such that there is oneidentification element from each of the at least three groups. Evenlarger numbers may be obtained by increasing the numbers of members ineach group and/or the numbers of groups present. In addition, the numberof members of each group may be the same, or different in some cases.

It should be noted that in other embodiments, other coding methods arealso possible. For instance, the distinguishable elements may be used torepresent binary digits, such that the nucleic acid probes or othersamples are arbitrarily numbered and are identified by adding the binarydigits corresponding to the distinguishable identification elements thatare present.

Accordingly, in some embodiments, a species-containing droplet can beidentified by introducing to the species-containing droplet, one or moreidentification elements that have been arranged in such a manner.Relatively large numbers of fluidic droplets can each be identified. Forinstance, a collection of tens, hundreds, or thousands of fluidicdroplets, containing differing nucleic acid probes, may be identified byadding, to each of the droplets, three or four identification elementsthat have been determined in such a manner.

The composition of a plurality of droplets (e.g., fluidized, rigidified,and/or species-containing) will now be discussed. In some embodiments,droplets of the present invention comprise a precursor material, wherethe precursor material is capable of undergoing a phase change, e.g., toform a rigidified droplet or a fluidized droplet. Therefore, the dropletmay be rigidified or fluidized by causing the precursor material torigidify or fluidized. For instance, a droplet may contain a gelprecursor and/or a polymer precursor that can be rigidified to form arigidified droplet comprising a gel and/or a polymer. The rigidifieddroplet, in some cases, may also contain a fluid within the gel orpolymer.

The rigidified droplet may be substantially porous or substantiallynon-porous. In some aspects, the rigidified droplet will besubstantially porous such that at least one species may be containedinternally within the rigidified droplet. As used herein, “containedinternally” or “added internally” means that the species issubstantially surrounded by the droplet. In other embodiments, however,a species may be contained within a non-porous droplet, or the speciesmay be contained on the surface of the droplet (e.g., at an interfacebetween the droplet and the fluid surrounding the droplet).

A droplet may be caused to undergo a phase change using any suitabletechnique. For example, a rigidified droplet may form a fluidizeddroplet by exposing the rigidified droplet to an environmental change. Adroplet may be fluidized or rigidified by a change in the environmentaround the droplet, for example, a change in temperature, a change inthe pH level, change in ionic strength, exposure to a electromagneticradiation (e.g., ultraviolet light), addition of a chemical (e.g.,chemical that cleaves a crosslinker in a polymer), and the like. Someexamples are given below.

As a specific example, in some cases, a droplet may be caused to undergoa phase change by raising or lowering the temperature of the dropletfrom a first temperature to a second temperature. For example, a firsttemperature may be raised or lowered to a second temperature by at leastabout 5° C., at least about 10° C., at least about 15° C., at leastabout 20° C., at least about 22° C., at least about 25° C., at leastabout 30° C., at least about 35° C., at least about 40° C., at leastabout 50° C., at least about 60° C., at least about 70° C., at leastabout 80° C., at least about 90° C., at least about 100° C., or to anyother suitable temperature that may cause the droplet to undergo a phasechange. As an specific example, a fluidic droplet comprising agarose maybe rigidified by cooling the droplet to a temperature below the gellingtemperature of agarose, or a rigidified droplet comprising agarose maybe fluidized by warming. In some cases, the temperature change is chosenin part such that a species (e.g., a cell) contained within the dropletremains unchanged. Non-limiting examples of gels that may form upon achange in temperature include agarose, a PEG-PLGA-PEG triblockcopolymer, Matrigel, or the like.

As another example, a droplet may be caused to undergo a phase change byraising or lowering the pH of the droplet from a first pH to a secondpH. For example, a first pH may be raised or lowered to a second pH byat least about 0.5 pH units, at least about 1 pH unit, at least about 2pH units, at least about 3 pH units, at least about 4 pH units, at leastabout 5 pH units, at least about 6 pH units, at least about 8 pH units,at least about 10 pH units, at least about 14 pH units, or to any othersuitable pH that may cause the droplet to undergo a phase change. Insome cases, the pH of the droplet may be changed from acidic to basic,basic to acidic, less acidic to more acidic, more acidic to less acidic,more basic to less basic, less basic to more basic, and the like.Non-limiting examples of gels that may undergo a phase change upon achange in pH include cellulose acetate phthalate latex and cross-linkedpoly acrylic or other carbomer derivatives (e.g., Polycarbophil® andCarbopol®).

As yet another example, the droplet may be caused to undergo a phasechange by reaction with a chemical reagent, for example, a crosslinkingreagent. For example, a polymer contained within a liquid can becrosslinked, thereby turning the liquid into a solid or a gel state bycrosslinking the chains of the polymer together. In some instances, acrosslinking reaction may be initiated by heat, pressure, orelectromagnetic radiation. In certain cases, a crosslinking agent willbe used to rigidify a droplet. Addition of a cleaving reagent may causethe rigidified droplet to be fluidized (e.g., the cleaving agent willcause the crosslinks that formed during crosslinking to be cleaved).Examples of rigidified droplets that may be prepared using crosslinkingreagents are discussed more herein.

In some embodiments, a rigidified droplet may be a gel droplet (e.g., adroplet comprising or consisting essentially of a gel). As used herein,the term “gel” is given its ordinary meaning in the art and refers to amaterial comprising a polymer network that is able to trap and containfluids. For example, a rigidified droplet may contain fluid from thefluidic droplet prior to rigidification of the fluidic droplet. The gelmay comprise polymer chains that are crosslinked. The degree ofcrosslinking may be varied, in some cases, to tailor the extent to whichthe gel absorbs or retains fluids. Those of ordinary skill in the artwill be able to select appropriate materials suitable for use as gels.In some cases, a gel may be formed from a gel precursor. For instance,the gel precursor may comprise a material that forms a gel upon reactionwith another material (e.g., a photoinitiator or crosslinker). Anexample of a gel precursor includes polyacrylamide. In anotherembodiment, the gel precursor comprises a material that forms a gel uponapplication of electromagnetic radiation to the material, such aschitosan or poly(ethylene) glycol.

In some cases, a gel may be altered to form a fluidized state, e.g., afluidic droplet. For instance, a polymer droplet may be fluidized bycleaving the crosslinks formed in the gel. Different types of gels andgel precursors that can be used in accordance with the present inventionare described in more detail below.

In some embodiment, the gel is a natural gel; that is, abiologically-derived gel. A natural gel may include, for example,agarose (e.g., low melting point agarose), collagen, fibrin, laminin,Matrigel, alginate, and combinations thereof In one particularembodiment, agarose is used. Droplets comprising natural gels and gelprecursors, in some instances, may be rigidified or fluidized by achange in the temperature or pH of the droplet, etc.

Non-limiting examples of materials capable of forming gels from a liquidprecursor include, but are not limited to, silicon-containing polymers,polyacrylamides (e.g., poly(N-isopropylacrylamide)), crosslinkedpolymers (e.g., polyethylene oxide, polyAMPS and polyvinylpyrrolidone),polyvinyl alcohol, acrylate polymers (e.g., sodium polyacrylate), andcopolymers with an abundance of hydrophilic groups. Those of ordinaryskill in the art can choose appropriate polymers that can becrosslinked, as well as suitable methods of crosslinking, based upongeneral knowledge of the art in combination with the description herein.

In some embodiments, a gel droplet may comprise a sol-gel. The term“sol-gel” as used herein means a gel derived from a sol, either bypolymerizing the sol into an interconnected solid matrix, or bydestabilizing the individual particles of a colloidal sol by means of anexternal agent. In general, the sol-gel process involves the change of acolloidal suspension system into a gel phase exhibiting a significantlyhigher viscosity. In some cases, the first liquid may comprise a sol-gelprecursor comprising a mixture of solid particles (e.g., inorganicsalts) suspended in a liquid, where a series of reactions includinghydrolysis and polymerization reactions may be performed to form arigidified droplet (e.g., a colloidal suspension). A non-limitingexample of a sol-gel is silica xerogel. In some cases, the gel may be anorganogel, where the polymer may be swollen by addition of an organicsolvent.

A variety of definitions are now provided which will aid inunderstanding various aspects of the invention. Following, andinterspersed with these definitions, is further disclosure that willmore fully describe the invention.

A “droplet,” as used herein, is an isolated portion of a first fluidthat is completely surrounded by a second fluid. It is to be noted thata droplet is not necessarily spherical, but may assume other shapes aswell, for example, depending on the external environment. In oneembodiment, the droplet has a minimum cross-sectional dimension that issubstantially equal to the largest dimension of the channelperpendicular to fluid flow in which the droplet is located. Thediameter of a droplet, in a non-spherical droplet, is the diameter of aperfect mathematical sphere having the same volume as the non-sphericaldroplet. The fluidic droplets may be created using any suitabletechnique.

In some embodiments, a plurality of droplets may be prepared usingmicrofluidic techniques, such as those disclosed in U.S. patentapplication Ser. No. 11/024,228, filed Dec. 28, 2004, entitled “Methodand Apparatus for Fluid Dispersion,” by Stone, et al., published as U.S.Patent Application Publication No. 2005/0172476 on Aug. 11, 2005; U.S.patent application Ser. No. 11/246,911, filed Oct. 7, 2005, entitled“Formation and Control of Fluidic Species,” by Link, et al., publishedas U.S. Patent Application Publication No. 2006/0163385 on Jul. 27,2006; or U.S. patent application Ser. No. 11/360,845, filed Feb. 23,2006, entitled “Electronic Control of Fluidic Species,” by Link, et al.,published as U.S. Patent Application Publication No. 2007/0003442 onJan. 4, 2007, each incorporated herein by reference.

As used herein, a “fluid” is given its ordinary meaning, i.e., a liquidor a gas. A fluid cannot maintain a defined shape and will flow duringan observable time frame to fill the container in which it is put. Thus,the fluid may have any suitable viscosity that permits flow. If two ormore fluids are present, each fluid may be independently selected amongessentially any fluids (liquids, gases, and the like) by those ofordinary skill in the art, by considering the relationship between thefluids. The fluids may each be substantially miscible or substantiallyimmiscible. In some cases, two fluids can be selected to besubstantially immiscible within the time frame of formation of a streamof fluids, or within the time frame of reaction or interaction. Ininstances where the portions remain liquid for a significant period oftime then the fluids should be substantially immiscible. In instanceswhere, after contact and/or formation, the dispersed portions arerigidified, the fluids may need not be substantially immiscible. Thoseof ordinary skill in the art can select suitable substantially miscibleor substantially immiscible fluids, using contact angle measurements orthe like, to carry out the techniques of the invention.

As used herein, a first entity is “surrounded” by a second entity if aclosed loop can be drawn around the first entity through only the secondentity. A first entity is “completely surrounded” if closed loops goingthrough only the second entity can be drawn around the first entityregardless of direction. A first entity is “substantially surrounded” ifthe loops going through only the second entity can be drawn around thefirst entity depending on the direction (e.g., in some cases, a looparound the first entity will comprise mostly of the second entity by mayalso comprise a third entity, or a fourth entity, etc.) In some aspectof the invention, the entities can both be fluids. For example, ahydrophilic liquid may be suspended in a hydrophobic liquid, ahydrophobic liquid may be suspended in a hydrophilic liquid, a gasbubble may be suspended in a liquid, etc. Typically, a hydrophobicliquid and a hydrophilic liquid are substantially immiscible withrespect to each other, where the hydrophilic liquid has a greateraffinity to water than does the hydrophobic liquid. Examples ofhydrophilic liquids include, but are not limited to, water and otheraqueous solutions comprising water, such as cell or biological media,ethanol, salt solutions, etc. Examples of hydrophobic liquids include,but are not limited to, oils such as hydrocarbons, silicon oils,fluorocarbon oils, organic solvents etc.

A method of the present in invention may provide a plurality ofdroplets. In some embodiments, the plurality of droplets contain a firstfluid and are substantially surrounded by a second fluid. In most, butnot all embodiments, the first fluid and the second fluid aresubstantially immiscible. In some cases, however, the first and secondfluids may be miscible. In some, but not all embodiments, the pluralityof the droplets may be produced using microfluidic techniques, asdiscussed more herein. “Microfluidic,” as used herein, refers to adevice, apparatus or system including at least one fluid channel havinga cross-sectional dimension of less than 1 mm, and a ratio of length tolargest cross-sectional dimension of at least about 3:1.

As discussed herein, droplets produced using a microfluidic device mayallow for the production of a plurality of droplets which havesubstantially the same composition. As used herein “substantially thesame composition” refers to at least two droplets which compriseessentially the same material (e.g., fluid, polymer, gel, etc.). In somecases, two droplets which have substantially the same composition differin their composition by no more than about 0.5%, no more than about 1%,no more than about 2%, no more than about 3%, no more than about 4%, nomore than about 5%, no more than about 10%, no more than about 20%, andthe like. Two droplets which have substantially the same composition mayonly differ in their composition because they contain distinguishablespecies. For example, the first droplet may comprise of a first fluidand contain internally at least one first species and the seconddroplets may comprise the same first fluid and contain internally atleast one second species, where the first species and the second speciesare distinguishable with respect to each other. That is, the dropletcomprise the same material (e.g., they are both formed of the samefluid, polymer, gel, etc.) but they each comprise at least onedistinguishable species with respect to the other species.

The plurality of fluidic droplets (e.g., prepared using a microfluidicdevice) may be polydisperse (e.g., having a range of different sizes),or in some cases, the fluidic droplets may be monodisperse orsubstantially monodisperse, e.g., having a homogenous distribution ofdiameters, for instance, such that no more than about 10%, about 5%,about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have anaverage diameter greater than about 10%, about 5%, about 3%, about 1%,about 0.03%, or about 0.01% of the average diameter. The “averagediameter” of a population of droplets, as used herein, is the arithmeticaverage of the diameters of the droplets. Those of ordinary skill in theart will be able to determine the average diameter of a population ofdroplets, for example, using laser light scattering or other knowntechniques. As non-limiting examples, the average diameter of a dropletmay be less than about 1 mm, less than about 500 micrometers, less thanabout 200 micrometers, less than about 100 micrometers, less than about75 micrometers, less than about 50 micrometers, less than about 25micrometers, less than about 10 micrometers, or less than about 5micrometers. The average diameter of the droplet may also be at leastabout 1 micrometer, at least about 2 micrometers, at least about 3micrometers, at least about 5 micrometers, at least about 10micrometers, at least about 15 micrometers, or at least about 20micrometers in certain cases.

In some, but not all embodiments, all components of the systems andmethods described herein are microfluidic. “Microfluidic,” as usedherein, refers to a device, apparatus or system including at least onefluid channel having a cross-sectional dimension of less than 1 mm, anda ratio of length to largest cross-sectional dimension perpendicular tothe channel of at least about 3:1. A “microfluidic channel,” as usedherein, is a channel meeting these criteria. As mentioned above, aplurality of droplets of the present invention may be produced usingmicrofluidic techniques. A benefit of using a plurality of dropletsprepared using microfluidic techniques to prepare a plurality offluidized or rigidified droplets comprising species is that thedroplets, in most cases, will be monodisperse. Therefore, the propertiesof the droplets (e.g., size, shape, composition) of a first droplet inthe suspension comprising a first type of species should besubstantially similar to a second droplet in the suspension comprising asecond type of species (with the exception of the distinguishingspecies). The monodisperse property of this technique may be difficultto achieve using other techniques, for example, producing a first and asecond droplet comprising a first and a second species, respectively,directly from a microfluidic instrument (as opposed to the addition ofthe species after formation of a plurality of substantially similardroplets).

Microfluidic systems may be provided that are able to cause two or moredroplets to fuse or coalesce into one droplet, for example, in caseswhere the two or more droplets ordinarily are unable to fuse orcoalesce, for example due to composition, surface tension, droplet size,etc. as known to those of ordinary skill in the art. Examples ofembodiments in which two or more droplets are fused have been describedabove. The fluidic droplets may be fused together using any suitabletechnique, for example, as discussed in U.S. patent application Ser. No.11/246,911, filed Oct. 7, 2005, entitled “Formation and Control ofFluidic Species,” by Link, et al., published as U.S. Patent ApplicationPublication No. 2006/0163385 on Jul. 27, 2006; or U.S. patentapplication Ser. No. 11/360,845, filed Feb. 23, 2006, entitled“Electronic Control of Fluidic Species,” by Link, et al., published asU.S. Patent Application Publication No. 2007/0003442 on Jan. 4, 2007,each incorporated herein by reference. As an example, in microfluidicsystems, the surface tension of the droplets, relative to the size ofthe droplets may prevent fusion or coalescence of the droplets fromoccurring. In one embodiment, two droplets may be given oppositeelectrical charges (i.e., positive and negative charges, not necessarilyof the same magnitude), which may increase the electrical interaction ofthe two droplets such that fusion or coalescence of the droplets canoccur. Electrical charges (positive or negative) may be imparted ontodroplets through the use of Taylor cones, or through any other suitabletechniques. For instance, an electric field may be imposed on a reactorcontaining the droplets, the droplets may be passed through a capacitor,a chemical reaction may occur to cause the droplets to become charged,flowing the droplets over a region with opposite wetting properties,etc.

The “cross-sectional dimension” of the channel is measured perpendicularto the direction of fluid flow. Most fluid channels in components of theinvention have maximum cross-sectional dimensions less than 2 mm, and insome cases, less than 1 mm. In one set of embodiments, all fluidchannels containing embodiments of the invention are microfluidic orhave a largest cross sectional dimension of no more than 2 mm or 1 mm.

In another embodiment, the fluid channels may be formed in part by asingle component (e.g., an etched substrate or molded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids in bulk and to deliver fluids to components of the invention. Inone set of embodiments, the maximum cross-sectional dimension of thechannel(s) containing embodiments of the invention are less than 500microns, less than 200 microns, less than 100 microns, less than 50microns, or less than 25 microns.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square, or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least about 2:1, more typically at least about 3:1, atleast about 5:1, or at least about 10:1 or more. An open channelgenerally will include characteristics that facilitate control overfluid transport, e.g., structural characteristics (an elongatedindentation) and/or physical or chemical characteristics (hydrophobicityvs. hydrophilicity) or other characteristics that can exert a force(e.g., a containing force) on a fluid. The fluid within the channel maypartially or completely fill the channel. In some cases where an openchannel is used, the fluid may be held within the channel, for example,using surface tension (i.e., a concave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.In some cases the dimensions of the channel may be chosen such thatfluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, positioned to intersect witheach other, etc.

Non-limiting examples of microfluidic systems that may be used with thepresent invention are disclosed in U.S. patent application Ser. No.11/246,911, filed Oct. 7, 2005, entitled “Formation and Control ofFluidic Species,” published as U.S. Patent Application Publication No.2006/0163385 on Jul. 27, 2006; U.S. patent application Ser. No.11/024,228, filed Dec. 28, 2004, entitled “Method and Apparatus forFluid Dispersion,” published as U.S. Patent Application Publication No.2005/0172476 on Aug. 11, 2005; U.S. patent application Ser. No.11/360,845, filed Feb. 23, 2006, entitled “Electronic Control of FluidicSpecies,” published as U.S. Patent Application Publication No.2007/000342 on Jan. 4, 2007; International Patent Application No.PCT/US2006/007772, filed Mar. 3, 2006, entitled “Method and Apparatusfor Forming Multiple Emulsions,” published as WO 2006/096571 on Sep. 14,2006; U.S. patent application Ser. No. 11/368,263, filed Mar. 3, 2006,entitled “Systems and Methods of Forming Particles,” published as U.S.Patent Application Publication No. 2007/0054119 on Mar. 8, 2007; U.S.Provisional Patent Application Ser. No. 60/920,574, filed Mar. 28, 2007,entitled “Multiple Emulsions and Techniques for Formation”; andInternational Patent Application No. PCT/US2006/001938, filed Jan. 20,2006, entitled “Systems and Methods for Forming Fluidic DropletsEncapsulated in Particles Such as Colloidal Particles,” published as WO2006/078841 on Jul. 27, 2006, each incorporated herein by reference.

The term “determining,” as used herein, generally refers to the analysisor measurement of a target analyte molecule, for example, quantitativelyor qualitatively, or the detection of the presence or absence of atarget analyte molecule. “Determining” may also refer to the analysis ormeasurement of an interaction between at least one species and a targetanalyte molecule, for example, quantitatively or qualitatively, or bydetecting the presence or absence of the interaction. Example techniquesinclude, but are not limited to, spectroscopy such as infrared,absorption, fluorescence, UV/visible, FTIR (“Fourier Transform InfraredSpectroscopy”), or Raman; gravimetric techniques; ellipsometry;piezoelectric measurements; immunoassays; electrochemical measurements;optical measurements such as optical density measurements; circulardichroism; light scattering measurements such as quasielectric lightscattering; polarimetry; refractometry; or turbidity measurements.

In one embodiment, a kit may be provided, containing one or more of theabove compositions. A “kit,” as used herein, typically defines a packageor an assembly including one or more of the compositions of theinvention, and/or other compositions associated with the invention, forexample, as previously described. Each of the compositions of the kitmay be provided in liquid form (e.g., in solution), in solid form (e.g.,a dried powder), etc. A kit of the invention may, in some cases, includeinstructions in any form that are provided in connection with thecompositions of the invention in such a manner that one of ordinaryskill in the art would recognize that the instructions are to beassociated with the compositions of the invention. For instance, theinstructions may include instructions for the use, modification, mixing,diluting, preserving, administering, assembly, storage, packaging,and/or preparation of the compositions and/or other compositionsassociated with the kit. The instructions may be provided in any formrecognizable by one of ordinary skill in the art as a suitable vehiclefor containing such instructions, for example, written or published,verbal, audible (e.g., telephonic), digital, optical, visual (e.g.,videotape, DVD, etc.) or electronic communications (including Internetor web-based communications), provided in any manner.

Incorporated herein by reference is a U.S. Provisional PatentApplication Ser. No. 61/098,674, filed Sep. 19, 2008, entitled “Creationof Libraries of Droplets and Related Species,” by Weitz, et al., U.S.Provisional Patent Application Ser. No. 61/008,862, filed Dec. 21, 2007,entitled “Systems and Methods for Nucleic Acid Sequencing,” by Weitz, etal., U.S. Provisional Patent Application Ser. No. 61/098,710, filed Sep.19, 2008, entitled “Systems and Methods for Nucleic Acid Sequencing,” byWeitz, et al., and International Patent Application No.PCT/US2008/013912, filed Dec. 19, 2008, entitled “Systems and Methodsfor Nucleic Acid Sequencing,” by Weitz, et al.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLE 1

The following example describes the formation of a plurality ofrigidified droplets comprising a first group of rigidified dropletscomprising a first species and a second group of rigidified dropletscomprising a second species, wherein the first species and the secondspecies are distinguishable from each other, according to one embodimentof the present invention.

A plurality of rigidified droplets were formed using microfluidictechniques as follows. A solution comprising water, 5% acrylamide, 0.25%bisacrylamide, 0.1% ammonium persulfate, and 1 uM of a firstacrydite-labeled DNA oligonucleotide 20-mer was flowed through thecenter channel of a microfluidic device and focused by oil containing0.1% TEMED in the side channels through a 10 micron nozzle to produceapproximately 12 micron pre-gel droplets, as depicted in FIG. 9A. Thedroplets were allowed to rigidify for 30 minutes at 50° C. viapolymerization reactions. FIG. 9B shows the plurality of rigidifieddroplets after being exposed to water.

The above steps (e.g., FIGS. 9A and 9B) were repeated for a second groupof droplets comprising a second acrydite-labeled DNA oligonucleotide20-mer that was distinguishable from the first DNA oligonucleotide20-mer. A suspension of droplets was formed comprising some of the firstgroup and the second group of rigidified droplets. The suspension ofrigidified droplets was exposed to a plurality of a first type and asecond type distinguishably labeled oligonucleotide specific to thefirst DNA oligonucleotide and the second DNA oligonucleotide,respectively. The distinguishably labeled oligonucleotides hybridized tothe specified DNA in the rigidified droplets. FIG. 9C shows afluorescence microscope image of the rigidified droplets followinghybridization wherein one type of rigidified droplets is shown in greyand the other type is shown in white.

While several embodiments of the invention have been described andillustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and configurations will depend upon the specificapplication or applications for which the teachings of the presentinvention is/are used. Those skilled in the art will recognize, or beable to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and/or claimed.The present invention is directed to each individual feature, system,material and/or method described herein. In addition, any combination oftwo or more such features, systems, articles, materials and/or methods,if such features, systems, articles, materials and/or methods are notmutually inconsistent, is included within the scope of the presentinvention.

All definitions as used herein are solely for the purposes of thisdisclosure. These definitions should not necessarily be imputed to othercommonly-owned patents and/or patent applications, whether related orunrelated to this disclosure. The definitions, as used herein, should beunderstood to control over dictionary definitions, definitions indocuments incorporated by reference, and/or ordinary meanings of thedefined terms.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“involving,” “holding,” and the like are to be understood to beopen-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedure, Section 2111.03.

1. A method, comprising: providing a plurality of droplets, each of theplurality of droplets comprising a first fluid and being surrounded by asecond fluid, wherein the first fluid and the second fluid aresubstantially immiscible; causing at least some of the plurality ofdroplets to undergo a phase change to form a plurality of rigidifieddroplets; exposing the plurality of rigidified droplets to a thirdfluid, wherein the third fluid is substantially miscible in the firstfluid; and adding at least one first species internally to at least someof the rigidified droplets. 2-3. (canceled)
 4. The method of claim 1,wherein the plurality of droplets are formed using microfluidictechniques. 5-7. (canceled)
 8. The method of claim 1, wherein theplurality of droplets comprise a gel precursor.
 9. (canceled)
 10. Themethod of claim 1, wherein the plurality of droplets are rigidified byraising or lowering the temperature of the droplet. 11-15. (canceled)16. The method of claim 1, wherein the third fluid comprises a pluralityof first species. 17-38. (canceled)
 39. The method of claim 1, furthercomprising the step of immobilizing the first species with respect tothe rigidified droplet it is internally contained in. 40-42. (canceled)43. The method of claim 1, wherein the phase change is from asubstantially liquid phase to a substantially gel phase. 44-72.(canceled)
 73. A method, comprising: providing a first plurality ofrigidified droplets, each rigidified droplet being surrounded by asecond fluid, the rigidified droplets being formed from a fluidicdroplet comprising a first fluid, wherein the second fluid issubstantially immiscible in the first fluid; exposing the firstplurality of rigidified droplets to a third fluid, wherein the thirdfluid is substantially miscible in the first fluid; adding at least onefirst species internally to at least some of the first plurality ofrigidified droplets to form a first group of species-containingdroplets; repeating the above acts with a second plurality of rigidifieddroplets and a second species to form a second group ofspecies-containing droplets; and forming a suspension comprising atleast some droplets of the first and second groups of species-containingdroplets. 74-121. (canceled)
 122. The method of claim 73, wherein therigidified droplets are fluidized prior to forming the plurality ofmicrofluidic droplets, such that each microfluidic droplet comprises atleast one fluidized droplet.
 123. The method of claim 122, wherein theplurality of microfluidic droplets are formed by fusing each of aplurality of fluidic droplet with at least one of the plurality offluidized droplets.
 124. The method of claim 73, wherein the pluralityof microfluidic droplets are formed by forming the droplets usingmicrofluidic techniques from a stream of fluid comprising the pluralityof rigidified droplets. 125-127. (canceled)
 128. The method of claim122, wherein the library of rigidified droplets are fluidized by achange in temperature. 129-134. (canceled)
 135. A method, comprising:providing a first plurality of rigidified droplets; immobilizing atleast one first species internally with respect to at least some of thefirst plurality of rigidified droplets to form a first group ofspecies-containing droplets; repeating the above acts with a secondplurality of rigidified droplets and a second species to form a secondgroup of species-containing droplets; and forming a suspensioncomprising at least some droplets of the first and second groups ofspecies-containing droplets. 136-148. (canceled)
 149. The method ofclaim 135, wherein the immobilization comprises the formation of a bond.150-155. (canceled)
 156. The method of claim 73, wherein the speciescontaining rigidified droplets are fluidized by agitation.
 157. Themethod of claim 135, wherein the first species is immobilized by sterichindrance